Anthropogenic disturbances are one of the primary factors that drive biodiversity loss. Temporal changes in biodiversity respond to such disturbances. In the present study, we investigated the temporal changes in taxonomic and functional diversity of fish assemblages in different habitats (Three Gorges Reservoir, TGR; running water above the TGR, UTGR; and tributary Chishui River, CSR) in the upper Yangtze basin after impoundment of the TGR from 2008 to 2015. During the survey, the taxonomic and functional composition of fish assemblages varied among the habitats. Although taxonomic diversity increased and functional diversity decreased significantly in the entire upper Yangtze basin, no significant changes in the diversities were observed in each habitat. The habitats showed directional changes in the composition of fish species. The slope of the directional changes in the TGR was more than that in the UTGR and CSR. These results indicated that the distribution of fish species was associated with the habitat after the impoundment. The assembly of the fish assemblage was driven primarily by the neutral process in the entire survey area but was promoted by a combination of species sorting and neutral process in each habitat. The impoundment caused more effects on the TGR than on the UTGR, but it slightly affected the CSR. Regarding fishing closure, conservation measures for fish diversity should be established according to the different habitats. The study findings suggest the removal of small dams in tributaries and ecological regulation in the mainstream and continuous monitoring of fish biodiversity.